Pipette tip and uses and methods thereof

ABSTRACT

The present invention provides a pipette tip, which can be used in in-vitro diagnostics, in particular in the diagnostic testing of body fluids, such as in coagulation testing. The Pipette tip contains two constituents in a spatially separated manner. The present invention furthermore provides a method of performing such diagnostics, e.g. coagulation analysis, and to the use of the pipette tip in such diagnostic testing.

The present invention relates to the field of in-vitro diagnostics, inparticular to the diagnostic testing of body fluids, such as bloodsamples. More specifically, the present invention relates to a pipettetip, which can be used in in-vitro diagnostics, in particular in thediagnostic testing of body fluids, such as in coagulation testing. Thepresent invention also relates to a method of performing suchdiagnostics, e.g. coagulation analysis, and to the use of the pipettetip in such diagnostic testing.

For many of these diagnostic methods, the body fluid has to be mixedwith other reagents to enable, or at least to improve, the diagnosticdetection of certain parameters to be measured. For example, incoagulation testing the patient's blood sample has to be combined withspecial “activator” reagents to simulate clotting processes. Inhemorheologic coagulation tests (also called “viscoelastic analysis”),the blood sample is mixed additionally with further coagulationmodifiers, such as platelet inhibitors, heparin inhibitors and/or lysisinhibitors.

The coagulation of blood is a highly complex process, starting withliquid blood and ending with the formation of a solid clot. It is animportant part of hemostasis, i.e. the cessation of blood loss from adamaged vessel, wherein a damaged blood vessel wall is covered by ablood clot to stop hemorrhage and aid repair of the damaged vessel.Disorders in the coagulation balance can lead to increased hemorrhageand/or thrombosis and embolism.

In a normal individual, coagulation is initiated within about 20 secondsafter an injury occurs to the blood vessel damaging the endothelialcells. Platelets immediately form a hemostatic plug at the site ofinjury. This process is called primary hemostasis. Secondary hemostasisfollows if plasma components called coagulation factors respond in acomplex cascade to finally form fibrin strands to strengthen theplatelet plug.

The coagulation cascade of secondary hemostasis has two pathways, theContact Activation pathway (formerly known as the Intrinsic Pathway) andthe Tissue Factor pathway (formerly known as the Extrinsic pathway) thatlead to fibrin formation. It was previously thought that the coagulationcascade consisted of two pathways of equal importance joined to a commonpathway. It is now known that the primary pathway for the initiation ofblood coagulation is the Tissue Factor pathway. The pathways are aseries of reactions, in which a zymogen of a serine protease and itsglycoprotein co-factor are activated to become active components, whichare then able to catalyze the next reaction in the cascade. Coagulationfactors are generally indicated by Roman numerals from I-XIII, with alowercase ‘a’ appended to indicate the activated form. Thereby, a fibrinclot is formed, which strengthens the platelet plug.

However, to avoid thrombosis and embolism, the formation of fibrin clotsis tightly controlled. The fibrin clot, i.e. the product of coagulation,is broken down in a process called fibrinolysis. Accordingly,fibrinolysis prevents blood clots from growing and becoming problematic.In fibrinolysis, the enzyme plasmin plays a major role, since plasmincuts the fibrin mesh at various places, leading to the production ofcirculating fragments that are cleared by other proteases and/or by thekidney and/or liver. Plasminogen is converted to active plasmin bytissue plasminogen activator (tPA) and urokinase, thereby allowingfibrinolysis to occur.

The detection of normal or decreased functionality of these coagulationand/or fibrinolysis components is important in order to assess patients'hemostasis disorders. If a hemostasis disorder is identified, a selectedtherapy can be applied for example to stop a bleeding.

Several methods of measuring the coagulation characteristics of bloodare known. Some such devices attempt to simulate the natural flow ofblood in the veins and arteries of a living subject, while othermeasurement techniques are performed in static blood volumes.

An accurate measurement of the ability of a patient's blood to coagulatein a timely and effective fashion is crucial to certain surgical andmedical procedures. Rapid and accurate detection of abnormalcoagulations is also of particular importance with respect toappropriate treatment to be given to patients suffering from clottingdisorders. Often the condition of such patients makes it necessary toadminister blood components, anti-coagulants, certain fibrinolyticagents, anti-platelet agents, or compounds inducing the reverse effectsof said agents. In these cases, the treatment dose can be adapted to theextent of a clotting disorder previously determined.

Measurements of blood clotting are provided by various devices, forexample as disclosed in U.S. Pat. No. 5,777,215 A and in U.S. Pat. No.6,537,819 B2. These devices measure the mechanical properties of theclot throughout its structural development. These systems are summarizedunder the term “viscoelastic methods”, as they continuously detectviscoelastic properties of the blood clot while its formation and lysis.Viscoelastic measurements of clotting blood are commonly also referredto as thromboelastography (TEG) measurements.

A number of references describe instruments for measuring blood clottingcharacteristics based upon mechanical movements. These instrumentsmonitor the elastic properties of blood as it is induced to clot under alow shear environment, i.e. in static blood volumes. The patterns ofchange in shear elasticity enable the determination of the kinetics ofclot formation, as well as the strength and stability of the formedclot. The strength and stability of the clot provide information aboutthe ability of the clot to perform the “work of hemostasis” (i.e., stopor prevent abnormal bleeding) and about the adequacy of bloodplatelet-fibrin interaction. The kinetics of clot formation mainlyprovides information about the functionality of coagulation factors.Analysis of all of this information provides results which are useful topredict bleeding, to monitor and manage thrombosis, or to monitorfibrinolysis.

Moreover, as the clotting process consists of various interlinkedcomponents, specific activators and inhibitors of the clotting processmay be applied in order to detect hemostasis disorders morespecifically. Such reagents useful in viscoelastic analysis may comprisean initial activator (e.g., an activator of either the intrinsic or theextrinsic pathway), one or more inhibitors (e.g., fibrinolysisinhibitors, heparin inhibitors, platelet inhibitors), one or morefurther specific factor(s) of the coagulation cascade, calcium (CaCl₂),phospholipids, and/or stabilizers.

Different reagent concepts for modified viscoelastic measurements aredescribed in the literature, including (i) ReoPro-modified TEG asdescribed in Wenker et al.: Thromboelastography, The Internet Journal ofAnesthesiology, 2000, Volume 1 Number 3,http://www.ispub.com/ostia/index.php?xmlFilePath=journals/ija/vol1n3/teg.xmland Ruttmann et al.: Hemodilution Enhanced Coagulation Is Not Due toPlatelet Clumping, Anesthesiology 2004; 101: A150; (ii) Recombiplastin-and ReoPro-modified TEG as described inhttp://www.transfusionguidelines.org.uk/docs/pdfs/bbt_app-use_teg-sop-example.pdf;and (iii) TF- and Trasylol-modified TEG as described in Tanaka et al.:Evaluation of a novel kallikrein inhibitor on hemostatic activation invitro, Thrombosis Research, Volume 113, Issue 5, 2004, Pages 333-339,whereby TF- and Trasylol-modified TEG is based on the combination ofcommercially available activator reagents intended for other tests, suchas the prothrombin time activator Innovin or Recombiplastin®, combinedwith customer-made CaCl₂ solution and drugs, such as ReoPro® (abciximab)and Trasylol® (aprotinin).

However, in those described concepts standardization is low and manycomplicated pipetting steps are included, resulting in many sources ofuser error.

There are other reagent systems on the market, which are based on avariety of reagents. For example, ROTEM® analysis (Manufacturer: TernInnovations GmbH, Munich, Germany) provides a reagent system forviscoelastic measurements, which is based on standardized reagents, mostof which are provided to the customer in a liquid form, which arepipetted by the user into the test cup using standardized operatingprocedures. This standardizes the application, however, it stillrequires several pipetting steps for the analysis. For example, toperform a platelet inhibited test together with an extrinsicallyactivated test, the pipetting of blood, CaCl₂ solution, extrinsicactivator and a platelet inhibitor may result in the performance of atotal of eight pipetting steps (including three times changing of thetip during one test procedure) and the need for three different reagentsthat have to be handled by the user. This provides a requirement fortraining, consumes time, and is a potential source of error.

Some of the further reagent systems on the market are liquid, and haveto be pipetted into a cup (e.g. CaCl₂ solution), some are provided indried form in the measurement cup (such as heparinase) and some areprovided in small vials, in a quantity intended for one test. Acharacteristic of these reagents is that still each reagent is typicallyprovided singly, and therefore several steps are required at least fortests requiring more than one active reagent.

To provide a simpler reagent system for viscoelastic measurements ofblood or blood components, the provision of stable liquid combinationsof the reagents in the working concentration was investigated. However,no such stable liquid combination could be achieved due to the mutualinteractions of the different substances while being mixed together fora longer period. Some components negatively affect the stability of eachother when kept mixed together in the liquid phase at higherconcentrations; for example, CaCl₂ disturbs the stability of TissueFactor reagent in liquid phase over the time. Moreover, if thesecombined reagents are provided in an amount sufficient for exactly onetest, another problem arises: the very small portion of a liquid reagentmight stick to parts of the reagent container or the cap and might thusnot mix sufficiently with the sample, i.e. the test liquid, when theanalysis is performed.

To avoid these problems, Kolde et al. disclosed in US 2004/0071604 A1 asystem providing freeze-dried reagents separately in their workingconcentrations for one test in a measurement cup (which receives thevolume of the sample during measurement). In particular, Kolde et al.disclose a cup system for viscoelastic analyses, in which the lower endof the cup is divided in several sections or ‘reagent chambers’. Thisallows to place the reagents independently into the different chambers,without mixing them and then to freeze-dry the reagents.

However, disadvantages of this solution include the need for a veryprecise pipetting process, as the separate reagent chambers are verysmall (<5 mm diameter). Another problem is that the reagent drops might‘jump’ out of their section as induced by vibrations in the reagentfilling line and mix with each other. A further problem is possibleair-drying of the small reagent drops during the processing under roomconditions before the lyophilization process begins.

Calatzis et al. disclosed in US 2010/190193 A1 another option byproviding freeze-dried reagents all mixed together in their workingconcentrations for one test in a measurement cup or in a standardreagent container. Thereby, it is suggested that all reagents areco-lyophilized in one reagent container or directly in the measurementcup.

This approach, however, can induce instabilities and variances in theproduction process due to mixing of all reagents and resulting mutualinteraction during the freezing process. Instabilities can also beinduced during the freezing process due to corresponding well-knownchanges in the pH conditions of the reagent mix. Accordingly, Calatziset al. suggested to stabilize the production process by diluting thereagent mix well below the concentration that is required inviscoelastic testing and compensate for the lower reagent content byproportionally increasing the lyophilized volume. But since the costlyfreeze-drying process is disproportionally prolonged by such volumeincreases, this approach reduces the production efficiency considerably.Moreover, co-lyophilized formulations can be substantially less stablethan the separated components depending on the residual moisture in thelyophilized reagent compound, which requires even longer processing timeduring production.

One further shortcoming of the systems disclosed in US 2004/0071604 A1and in US 2010/190193 A1 is that protein stabilizers are required thatcan later interfere with the adhesion strength of the blood clot on thecup surface during the viscoelastic measurement.

To overcome the above-mentioned problems, Schubert et al. disclosed afurther option in US 2013/102015 A1, where each reagent is dilutedseparately in an excipient solution and lyophilized in the form of smallpellets made of the excipient framework. This approach keeps thereagents apart during the whole production process as well as duringstorage and minimizes in this way all mutual interactions. On the otherhand, an additional component—the excipient—has to be added and must beextensively verified for eventual interference with the coagulationcharacteristics. Besides this, the process of pellet production becomesconsiderably more costly than liquid dispensing. It requires highlyindividual equipment for both, pellet production and later pelletdistribution into reagent containers or measurement cups.

In view of the above, it is the object of the present invention toovercome the drawbacks of current reagent systems for viscoelasticanalysis outlined above and to provide a pipette tip, which can be usedfor diagnostic testing such as viscoelastic analysis, as well asrespective methods, which are simplifying diagnostic methods such asviscoelastic analysis, for example by minimizing the number of pipettingsteps. Moreover, it is also an object of the present invention toprovide a pipette tip, which can be used in viscoelastic analysis,wherein the required reagent composition has an improved long-termstability but does not require costly manufacturing equipment oradditional (excipient) materials in the reagent composition. It is alsoan object of the present invention to provide a pipette tip forviscoelastic analysis, and methods and uses thereof which allow for asafe, reproducible and easy to use procedure for different tests. It isalso an object of the present invention to provide a pipette tip, whichcan be used in viscoelastic analysis, and relating methods, whichrequire only standard filling and drying procedures during productionwithout individually specialized and costly automation equipment,thereby allowing cost-saving production. It is a further object of thepresent invention to provide a diagnostic method, which providesreliable and reproducible results, is easy to handle and which providesa standardized system for the determination of the coagulationcharacteristics of a blood sample.

The above objects are achieved by means of the subject-matter set outbelow and in the appended claims.

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isnot intended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention, the term “comprise” encompasses the term “consistof”. The term “comprising” thus encompasses “including” as well as“consisting” e.g., a composition “comprising” X may consist exclusivelyof X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

Pipette Tip

In a first aspect the present invention provides a pipette tipcomprising a constituent (A) and a constituent (B) in a spatiallyseparated manner, wherein the constituents (A) and (B) are adapted toform a diagnostic composition upon combination.

In other words, the constituents (A) and (B) are adapted to form adiagnostic composition upon combination of the constituents (A) and (B),i.e. the diagnostic composition corresponds in particular to acombination of the constituents (A) and (B). Thus, the componentscomprised by the diagnostic composition are comprised by one or both ofthe constituents (A) and (B). Preferably the diagnostic compositioncomprises at least the following components (i) and (ii):

-   -   i) an activator of coagulation; and    -   ii) a calcium salt.

As used herein, a “pipette tip” is the tip of a pipette. A pipette is alaboratory tool commonly used to transport a measured volume of liquid.Pipettes come in several designs for various purposes with differinglevels of accuracy and precision, from single piece glass pipettes tomore complex adjustable or electronic pipettes. Many pipette types workby creating a partial vacuum above the liquid-holding chamber andselectively releasing this vacuum to draw up and dispense liquid.Measurement accuracy varies greatly depending on the style.

Preferably, the pipette is an air-displacement micropipette, which is atype of adjustable micropipette that measures volumes between about 0.1μl-1000 μl (1 ml). These pipettes require disposable tips that come incontact with the fluid. The four standard sizes of micropipettescorrespond to four different disposable tip colors:

-   (1) Pipette “P10” for pipetting a volume of 0.5-10 μl, whereby the    corresponding tips are usually of white color;-   (2) Pipette “P20” for pipetting a volume of 2-20 μl, whereby the    corresponding tips are usually of yellow color;-   (3) Pipette “P200” for pipetting a volume of 20-200 μl, whereby the    corresponding tips are usually of yellow color; and-   (4) Pipette “P1000” for pipetting a volume of 200-1000 μl, whereby    the corresponding tips are usually of blue color.

Accordingly, the pipette tip according to the present invention ispreferably a disposable pipette tip, more preferably a disposablepipette tip for an air-displacement micropipette, even more preferably adisposable pipette tip for an air-displacement micropipette forpipetting a volume of 5-1000 microliter.

It is also preferred that the pipette tip is the tip of any otherpipette, for example the tip of a positive displacement pipette,preferably a disposable tip of a positive displacement pipette, which isin particular a microsyringe (plastic), composed of a plunger whichdirectly displaces the liquid; the tip of a volumetric pipette, the tipof a graduated pipette, or any other type of pipette. Thereby, it ispreferred that it is a disposable pipette tip.

In general, the pipette tip according to the present invention, whichcontains constituents (A) and (B) of the diagnostic compositioncomprises:

-   -   an open upper end fitting to the corresponding end of a pipette;        and    -   an open lower end.

The “upper” and “lower” part/end of a pipette tip, as used herein, referto the (ideal) orientation of the pipette tip in practice: Since it isdesirable that the liquid to be pipetted is located only in the lowestpart of the pipette tip (to avoid contamination of the pipette and toavoid air bubbles impairing the measurement accuracy of the volume to bepipetted), a pipette is preferably held perpendicular (to the earth'ssurface). Accordingly, the liquid to be pipetted typically enters thepipette tip via its open lower end (during aspirating) and is alsoreleased via the pipette tip's open lower end. The open upper end, incontrast, fits to the corresponding (lower) end of a pipette and is usedto fix the pipette tip on the pipette.

Preferably, the open upper end of the pipette tip and the open lower endof the pipette tip have a circular shape. It is also preferred that theopen lower end of the pipette tip has a smaller diameter than the openupper end.

Preferably, the pipette tip according to the present invention furthercomprises

-   -   at least one region of the inner surface of the pipette tip        containing one (single) of the constituents (A) and (B), in        particular wherein the constituent (A) or (B) is directly        deposited on said inner surface of the pipette tip (i.e. without        any further layers/substances between the at least one of the        constituents (A) and (B) and said inner surface of the pipette        tip; and/or    -   at least one insert containing one (single) of the        constituents (A) and (B), wherein the insert preferably        comprises a homogenously porous structure, preferably of a        natural or artificial polymer.

Preferably, the pipette tip according to the present invention comprisesone region of the inner surface of the pipette tip as described abovecontaining constituent (A) and one region of the inner surface of thepipette tip as described above containing constituent (B). Preferably,the pipette tip according to the present invention comprises one regionof the inner surface of the pipette tip as described above containingconstituent (A) and one insert as described above containing constituent(B). Preferably, the pipette tip according to the present inventioncomprises one insert as described above containing constituent (A) andone region of the inner surface of the pipette tip as described abovecontaining constituent (B). Preferably, the pipette tip according to thepresent invention comprises one insert as described above containingconstituent (A) and one insert as described above containing constituent(B). More preferably, the pipette tip according to the present inventioncomprises (i) one region of the inner surface of the pipette tip asdescribed above containing constituent (A) and one insert as describedabove containing constituent (B) or (ii) one insert as described abovecontaining constituent (A) and one region of the inner surface of thepipette tip as described above containing constituent (B). Mostpreferably, the pipette tip according to the present invention comprises(i) one region of the inner surface of the pipette tip as describedabove containing constituent (A) and one insert as described abovecontaining constituent (B).

Thereby, constituents (A) and (B) are kept for the (allowed) storagetime of the tip.

Common pipetting tips are usually produced by injection molding oftransparent polymers like polyethylene, polypropylene, polycarbonate,and many others. Accordingly, the pipette tip is preferably made ofplastics, such as polyethylene, polypropylene and/or polycarbonate. Somepipette tips are slightly colored by adding a dye to the raw materialand this coloring refers to a certain size of the corresponding pipette(e.g., blue for 1000 μl pipettes and yellow for 100 μl pipettes).Preferably the pipette tip is molded, more preferably having a coloring(transparent or opaque). Such a color is preferably used to indicate theconstituents forming a diagnostic composition upon combination and,thus, to the diagnostic test that can be performed by using this tip.

In the pipette tip, constituents (A) and (B) are present in a spatiallyseparated manner. In other words, in the pipette tip constituents (A)and (B) are not in contact with each other. Such spatial separation thusenables that constituents (A) and (B) are not in contact with eachother, thereby avoiding undesired chemical reactions of constituents (A)and (B).

Preferably, the pipette tip comprises

-   -   a) a compartment (a) containing the constituent (A); and/or    -   b) a compartment (b) containing the constituent (B).

The term “compartment” as used herein refers to a separate part insidethe pipette tip. In other words, constituent (A) is preferably containedin a separate part of the pipette tip and constituent (B) is preferablycontained in another separate part of the pipette tip. Preferably, acompartment is structurally confined, e.g. by its structure itself (forexample if the compartment is an insert, such as a porous insert), by(side) walls, protrusions, membranes or other confining structures. Itis also preferred that a compartment is not (completely) structurallyconfined, such as distinct compartments in the pipette tip beingdistinct sections of the pipette tip, such as horizontal (crosswise)sections of the pipette tip or longitudinal sections of the pipettetip—without (constructional) structures for separating one section fromanother.

Preferably, the compartments (a) and (b) are designed to prevent anycontact between constituents (A) and (B), in particular as long as theyare in liquid form, e.g. during and/or after the manufacturing/fillingprocess of the tip.

Preferably, the compartments are designed to enable filling withdifferent volumes. In other words, the volumes of the compartments (a)and (b) are preferably different from each other, i.e. the volume ofcompartment (a) is preferably larger or smaller than the volume ofcompartment (b). For example, compartment (a) may be filled with lessthan 5 μl liquid constituent (A) and compartment (b) may be filled withmore than 5 μl liquid constituent (B), thereby implying that compartment(a) has a volume of less than 5 μl and compartment (b) has a volume ofmore than 5 μl. For example, compartment (a) may be filled with lessthan 2 μl liquid constituent (A) and compartment (b) may be filled withmore than 2 μl liquid constituent (B), thereby implying that compartment(a) has a volume of less than 2 μl and compartment (b) has a volume ofmore than 2 μl. For example, compartment (a) may be filled with lessthan 10 μl liquid constituent (A) and compartment (b) may be filled withmore than 10 μl liquid constituent (B), thereby implying thatcompartment (a) has a volume of less than 10 μl and compartment (b) hasa volume of more than 10 μl. Such volumes enable the preparation of theliquid constituents A and B in their respective optimum concentration,in particular for the intended (blood) sample volume. Accordingly, thedifferent reagent filling volumes might be dependent on the total volumeof the pipetting tip. For example, a pipette tip with for a (blood)sample volume of about 300 μl might have one compartment suitable forvolumes of more than 5 μl reagent and one compartment suitable for lessthan 5 μl reagent. For example, a pipette tip with for a (blood) samplevolume of about 100 μl might have one compartment suitable for volumesabove 2 μl reagent and one compartment suitable for volumes less than 2μl reagent. For example, a pipette tip for a (blood) sample volume ofabout 600 μl might have one compartment suitable for volumes above 10 μlreagent and one compartment suitable for volumes less than 10 μlreagent.

Preferably, at least one of compartments (a) and (b) is formed by a(porous) insert. Accordingly, the pipette tip preferably comprises atleast one (porous) insert. Preferably, constituent (A) is comprised by(exactly) one (porous) insert and/or constituent (B) is comprised by(exactly) one (porous) insert. If both, constituents (A) and (B) arecomprised by (porous) inserts, the (porous) insert comprisingconstituent (A) is preferably different from the (porous) insertcomprising constituent (B) in order to ensure spatial separation ofconstituents (A) and (B). More preferably, compartment (b) containingconstituent (B) is formed by a (porous) insert. The term “insert” asused herein refers to an element, which is not comprised by conventional(non-pre-filled/non-modified) disposable pipette tips. Typically, an“insert” is made of a material different from the material of thepipette tip itself. However, the insert may also be made of the samematerial as the pipette tip. The term “porous” refers to an inserthaving pores as described below. Preferably, the pores of the porousinsert have a minimum pore diameter of 2 μm and a maximum pore diameterof 2.0 mm depending on the inner diameter of the pipette tip in theregion where the porous insert is placed as described below.

Preferably, the shape of the pipette tip is adapted to receive aninsert, in particular a porous insert (herein also called “plug”),preferably in the lower part of the pipette tip. The (porous) insert haspreferably a cylindrical or spherical shape. To receive a (porous)insert, the pipette tip may have a conventional (merely conical) shapeor the shape of the tip may be modified. Preferably, the shape of thetip may be modified in comparison to the shape of a regular conicalpipette tip in a way that the insert can be made of a cylindrical shape.In particular, it is preferred that such a preferred modified pipettetip has as a barely conical, but nearly cylindrical shape, morepreferably a cylindrical shape, over at least 2 mm of its entire length.Such a modified pipette tip can receive a cylindrical plug, and aconical shape of the insert can be avoided. Cylindrical plugs are easierand cheaper to produce than conical inserts because they can be blankeddirectly from sheet material. It is also preferred that the pipette tipis not modified and has, thus, a conventional (merely conical) shape.

Preferably, the porous insert is made of a support material. A “supportmaterial” preferably allows absorption of the (initially) liquidconstituent (A) and/or (B), e.g. during the manufacturing/fillingprocess of the pipette tip. The term “support material” as used hereinrefers to a material “supporting” a constituent comprised by the pipettetip, in particular a material “supporting” constituent (A) orconstituent (B) comprised by the pipette tip as described herein. Inother words, a constituent, in particular constituent (A) or constituent(B), comprised by the pipette tip as described herein may be depositedon a support material. Accordingly, a support material preferablyprovides a support structure for a constituent, in particularconstituent (A) or constituent (B), comprised by the pipette asdescribed herein. Preferably, the support material is a foam, such as apolymeric foam, e.g. a natural or an artificial foam, such as forexample a polyether foam, a polyesther foam, a polystyrol foam, or apolyurethane foam. More preferably, the support material is an open-cellfoam (or sponge) structure, and, even more preferably, the foamstructure has a low variation in pore sizes because similar pore sizesand high material homogeneity allow for even distribution of the liquidreagent in the foam plug during the filling process. Preferably, thepore size, in particular the minimum diameter of the pores, of thesupport material is at least 2 μm, thereby (i) allowing all possiblyapparent components of a human sample liquid (such as blood thrombocytesor red cells; bacteria in urea, etc.) to pass through the porous insert(made of the support material) during sample aspiration, (ii) allowingthe sample to dispense without creating closures in the support materialand/or (iii) allowing the sample to dispense without overly reducing itsaspiration/dispensing speed (e.g., to more than 15 seconds per fullvolume of the tip). The maximum diameter of the pores does preferablynot exceed values of one third of the inner diameter of the pipette tipin the region, where the porous insert is placed, to enable proper fitand sufficient enhancement of the surface area (e.g., maximum porediameter of 0.3 mm for a tip having 0.9 mm inner diameter in the regionwhere the porous insert is placed, or maximum pore size of 2.0 mm for atip having 6.0 mm inner diameter in the region where the supportmaterial is placed).

The pore diameter (e.g. a maximum or minimum pore diameter), as usedthroughout the present description, can be measured by methodswell-known to the skilled person, in particular by microscopic imaging.Thereby, microscopy is preferably performed under standard conditions(temperature: 22° C. and absolute pressure: 101.325 kPa), in particularreflecting the pore diameter when the pipette tip is used. Standardcomputer software for microscopic imaging typically provides tools formeasurement taking the resolution into account. Another option fordetermining the pore diameter, which is, however, less preferred thanmicroscopic imaging, is flow-rate measurement and determination of thepore diameter by using, e.g, Darcy's law.

It is also preferred that the foam-like material has a minimumelasticity modulus of >0.3 N/m² to enable simple fixation of apredominantly axially symmetrical (e.g., cylindrical or spherical)porous insert, such as a “foam plug”, (support material) within the tipby actively pressing it into a region where the inner diameter of thetip is smaller than the outer diameter of the plug and the resultingdecompression force keeping the plug in place. Additionally, saidminimum elasticity would make the handling of the foam plug duringmanufacturing easier when compared to materials that react with moreinelastic deformation to retention forces, acceleration forces, andother forces that are typical for item handling in automatedmanufacturing.

The elasticity modulus (e.g. a maximum or minimum elasticity modulus),as used throughout the present description, can be measured by methodswell-known to the skilled person, in particular by simple materialelongation under tensile stress perpendicular to a defined crosssection.

It is further preferred that at least one of compartments (a) and (b) ofthe pipette tip is formed by a porous insert made of support material,such as a foam plug, having a maximum elasticity modulus of <300 N/m²,which would enable reagent filling by using the suction forces asresulting from compression and subsequent decompression of the foam(sponge principle). The compression forces could be applied directly bythe employed filling means, e.g., a dispensing needle, or by additionalmeans.

Of note, the elasticity modulus values described above are to bemeasured for a macroscopic part of the foam material, in particular asaverage over a number of polymer walls and enclosed empty spheres (thepores), while the polymer itself might have a considerably largerelasticity modulus when processed differently than with foam extrusion.

In more general, the (porous) insert is preferably made of a plasticmaterial that can be injection-molded, sintered, extruded or foamed withthe formation of a rather homogeneous porosity of 2-500 μm pore sizes(minimum pore diameter: 2 μm and maximum pore diameter: 500 μm).Examples of such a plastic material, which is the preferred material ofthe (porous) insert, include polyethylene, polypropylene, polycarbonate,polyether, polyester, and the like. It is also preferred that the insertis not made of a material containing glass or metal, e.g. sheet metaland/or aluminum foil, more preferably the insert does not contain anyglass or metal, e.g. sheet metal and/or aluminum foil. It is alsopreferred that the pipette tip is not made of a material containingglass or metal, e.g. sheet metal and/or aluminum foil, more preferablythe pipette tip does not contain any glass or metal, e.g. sheet metaland/or aluminum foil.

Preferably, at least one of compartments (a) and (b) is formed by a(longitudinal or crosswise) section of the pipette tip comprising areagent layer. Accordingly, the pipette tip preferably comprises atleast one reagent layer. Preferably, constituent (A) is comprised by(exactly) one reagent layer and/or constituent (B) is comprised by(exactly) one reagent layer. If both, constituents (A) and (B) arecomprised by reagent layers, the reagent layer comprising constituent(A) is preferably different from the reagent layer comprisingconstituent (B) in order to ensure spatial separation of constituents(A) and (B) (in other words, the reagent layer comprising constituent(A) has preferably no contact with the reagent layer comprisingconstituent (B) in the pipette tip). More preferably, compartment (a)containing constituent (A) is formed by a (longitudinal or crosswise)section of the pipette tip comprising a reagent layer. To obtain areagent layer, the respective constituent (A) or (B) (or its components)is preferably deposited onto the inner surface of the pipette tip. Forexample, the respective constituent (A) or (B) (or its components) maybe deposited onto the inner surface of the pipette tip in its liquidform and thereafter the respective constituent (A) or (B) (or itscomponents) is/are dried, e.g. by lyophilization, heat-drying etc. Forexample, the respective constituent (A) or (B) (or its components) maybe sprayed in micro-drops (preferably drops having a diameter of no morethan 100 μm when sprayed onto the inner surface of the tip). Thereby,reagent clustering along the open lower end of the tip (and clogging ofthat end) is prevented. This furthermore allows wetting of the sidewallsand increases the covered surface, resulting in less reagent layerthickness and corresponding faster dissolution after sample adding.

Preferably, the pipette tip comprises a reagent layer having an eventhickness. Preferably, the reagent layer is located at the lowest partof the pipette tip, e.g. within the lowest third of the pipette tip(referring to the pipette tip's volume), preferably within the lowestquarter of the pipette tip (referring to the pipette tip's volume), morewithin the lowest fifth of the pipette tip (referring to the pipettetip's volume), and most within the lowest sixth of the pipette tip(referring to the pipette tip's volume).

In general, the positions of the at least two compartments (a) and (b)in the pipette tip are variable, but are preferably in the range wherethe sample liquid (in an amount as required to perform the diagnostictest) can completely wet the corresponding region. Thus, the position ofcompartments (a) and (b) within the tip is preferably within the lowerhalf of the pipette tip, more preferably within the lowest third of thepipette tip, even more preferably within the lowest quarter (fourth) ofthe pipette tip, whereby the terms “half”, “third” and “quarter(fourth)” refer to the respective volume in relation to the total volumeof the pipette tip, i.e. ½ of the total volume, ⅓ of the total volume or¼ of the total volume.

Preferably, the pipette tip comprises at least one (porous) insert asdescribed herein and at least one reagent layer as described herein.Thereby, it is preferred that the porous insert comprises constituent(A) and the reagent layer comprises constituent (B) or the porous insertcomprises constituent (B) and the reagent layer comprises constituent(A). If the pipette tip comprises at least one (porous) insert asdescribed herein and at least one reagent layer as described herein, (i)the at least one (porous) insert as described herein may be locatedabove the at least one reagent layer as described herein (referring toan orientation of the pipette tip as described above, in the context ofthe “upper and lower part/end” of the pipette tip), or (ii) the at leastone (porous) insert as described herein may be located below the atleast one reagent layer as described herein (referring to an orientationof the pipette tip as described above, in the context of the “upper andlower part/end” of the pipette tip). Preferably, the at least one(porous) insert as described herein may be located above the at leastone reagent layer as described herein (referring to an orientation ofthe pipette tip as described above, in the context of the “upper andlower part/end” of the pipette tip).

Preferably, at least the compartment having the larger volume asdescribed above is formed by a (porous) insert, e.g. made of a supportmaterial. For example, compartment (b) containing the constituent (B) isformed by a (porous) insert, e.g. made of a support material, orcompartment (a) containing the constituent (A) is formed by a (porous)insert, e.g. made of a support material.

Preferably, the pipette tip according to the present inventioncomprises:

-   -   a) a compartment (a) containing constituent (A) and not        containing the constituent (B); and    -   b) a compartment (b) containing constituent (B) and not        containing the constituent (A).

Thereby, it is more preferred that the constituents (A) and (B) areadapted to form a diagnostic composition upon combination, wherein thediagnostic composition (i.e. the constituents (A) and/or (B)) comprisesthe following components (i) and (ii):

-   -   i) an activator of coagulation; and    -   ii) a calcium salt.

Preferably, in the pipette tip according to the present invention asdescribed above, the constituent (A) is different from the constituent(B). In other words, constituents (A) and (B) are preferably not thesame.

Since the constituents (A) and (B) are adapted to form a diagnosticcomposition upon combination, wherein the diagnostic compositionpreferably comprises the components (i) and (ii), as described above,each of the constituents (A) and (B) is in particular a composition ofcomponents (or reagents) by itself. Preferably, constituent (A) is acomposition comprising component (i) or component (ii), and, optionally,further components. Accordingly, constituent (B) is preferably acomposition comprising component (i) or component (ii), and, optionally,further components. However, constituent (A) is preferably differentfrom constituent (B) and, thus, it is preferred that:

-   -   1) component (i) is comprised by constituent (A) and        component (ii) is comprised by constituent (B); or    -   2) component (ii) is comprised by constituent (A) and        component (i) is comprised by constituent (B).

An exemplary diagram showing a typical coagulation test, namely, aviscoelastic analysis (also referred to as viscoelastic measurement), isshown in FIG. 1 . The diagram curve represents the increasing clotfirmness in the measurement cup after an initial delay time (clottingtime). The curve develops until a maximum is reached that represents themaximum clot firmness of the sample.

By using the pipette tip according to the present invention, aviscoelastic analysis may be performed for example as follows:

-   -   1) a defined volume of a sample (e.g., whole blood, blood plasma        etc.) is aspirated into a pipette tip containing a        constituent (A) of the diagnostic composition in dry, wet, or        other formulation and a constituent (B) of the diagnostic        composition in dry, wet, or other formulation, thereby obtaining        a mixture of constituent (A), constituent (B) and the sample;    -   2) optionally, a short time delay (e.g., 0-30 s) allowing for        complete dissolution of constituents (A) and (B) of the        diagnostic composition within the sample liquid;    -   3) the mixture (or solution) of constituent (A), constituent (B)        and the sample is added directly into a measurement cup suitable        for viscoelastic measurement;    -   4) optionally, to improve the mixing of all components, the        resulting mixture is at least partially aspired again into the        pipette tip and subsequently released again into the measurement        cup (this step may be repeated one or more times); and    -   5) the viscoelastic measurement is started (after placing the        cup in the right measurement position if this has not been done        before).

Optionally, the sample may be added to a container containing a furtherconstituent of the diagnostic composition after step 2) and aspiredagain after a short dissolution time (0-30 s) and before step 3).

Accordingly, the user needs only a minimum of two pipetting steps foreach test to perform, while in the liquid reagent system according tothe prior art up to eight steps are required. Moreover, no change of thepipette tip is necessary. Thus, the present pipette tip—for example forthe determination of coagulation characteristics of a blood sample—canbe handled easier, thereby decreasing the likelihood of errors, whichcan be due to an imprecise line of action by a (potentially lessexperienced) operator. Therefrom, further advantages may arise as, forexample, a higher reproducibility of the results to be achieved, andthus, a higher degree of standardization.

The sample to be tested by use of the pipette tip according to thepresent invention is preferably liquid. Accordingly, it is also referredto herein to a liquid sample as sample liquid or test liquid. Morepreferably the sample liquid is a biofluid (also referred to as bodyfluid), i.e. a fluid originating from an organism, in particular a fluidoriginating from a human or an animal. Even more preferably the sampleliquid is blood, preferably whole blood, or one or more of its elements,e.g. plasma and/or cells. Particularly preferably the sample is a humanblood sample and comprises (whole) blood and/or blood plasma.

Accordingly, the present invention is directed to a diagnosticcomposition and a pipette tip, which can be used in in-vitro diagnosticsof a sample, such as coagulation tests, in particular in theviscoelastic analysis of a sample. The diagnostic composition preferablycontains at least one activator of coagulation and a calcium salt, i.e.components (i) and (ii). Optionally, the diagnostic composition maycontain further components as described below, e.g. one or more otherinhibitors and/or coagulation components. According to the presentinvention, the components of the diagnostic composition are separatedwithin the pipette tip, in particular the components of the diagnosticcomposition are contained in at least two distinct compartments of thepipette tip. Since a pipette tip and, optionally, a measurement cup areanyway required in in-vitro diagnostics of a sample, such as coagulationtests, in particular in the viscoelastic analysis of a sample, noadditional reagent containers are necessary and a loss of diagnosticcomposition or one or more components thereof, which is due to pipettingfrom one additional reagent container into another reagent container,can be avoided. Moreover, the present invention allows to provide therequired diagnostic composition with improved long-term stability andincreased reconstitution time for the contained bio-molecules, butwithout the need for using costly manufacturing equipment or additional(excipient) materials in the reagent composition. In clinicalapplication of the disclosed pipette tip, the number of pipetting stepsis minimized. In particular, the pipette tip according to the presentinvention is adapted to one single analysis of a blood sample and has asuperior reagent stability regarding several prior art compositions. Theinventive concept is based on the separation of the required substances,e.g. into two different compartments.

Optionally, the pipette tip according to the present invention maypreferably contain one or more further compartments, e.g. 1, 2, 3, 4, or5 further compartments, in addition to the compartment (A) containing aconstituent (A) and compartment (b) containing a constituent (B). Suchan additional compartments preferably contain constituents other thanconstituents (A) and (B), which also contribute to the diagnosticcomposition formed upon combination of the constituents (A) and (B).

Optionally, one or more additional (reagent) components may be providedby another (reagent) container, such as a regular reagent vial or ameasurement cup, in addition to the pipette tip, thereby providing adiagnostic kit.

It is also preferred, however, that the pipetting tip according to thepresent invention comprises exactly two constituents (A) and (B), i.e.no more constituents in addition to constituents (A) and (B).

Preferably, the pipette tip, which contains constituent (A) and (B) (andany optional additional reagent container) contain in particular aconstituent of the diagnostic composition in an amount sufficient forperforming one single viscoelastic analysis of a sample, in particulartest liquid. In particular, the pipette tip, which contains aconstituents (A) and (B) (and any optional additional reagent container)can be filled with reagents in either liquid, dry, essentially dry orany other formulation.

The amount sufficient for performing one single viscoelastic analysis ofa sample, for example a blood sample, is that amount required for eachof the constituents when all constituents are in mixture (i.e. in the“diagnostic composition”), which provides the required concentration ofthe reagents in the final diagnostic analysis, e.g. viscoelasticanalysis, of the sample, e.g. of a blood sample. Therefore, it is notnecessary to further portion the diagnostic composition before or aftermixing, preferably dissolving, it in a liquid.

Preferably, the final working concentration of reagents is achieved bymixing, the constituents with the sample directly in the pipette tip,but not by mixing the constituents in an amount of liquid diluent in thepipette tip and bringing this solution together with the intended samplesubsequently.

Further, it is preferred that the mixing is achieved by dissolution ofthe constituents within the sample.

Accordingly, the present invention (1) allows a separation of certainreagent ingredients that influence each other, which increases thereagent stability; (2) allows the formulation of ingredients in eitherliquid, dry, essentially or other form depending on the their stabilityand/or stability needs (in particular, some of the ingredients aretypically used in huge excess so that partial degradation is notfalsifying the test results, some ingredients are typically incrediblystable even in liquid form, and only some ingredients are typically lessstable in liquid form and not used in excess and must therefore betreated more carefully); and (3) saves additional costs and materialwaste (i.e., reagent vials) by employing containers that are used anywayfor performing the diagnostic test, such as viscoelastic tests.

Accordingly, the present invention provides a unique combination of twoconstituents (A) and (B) arranged in a single pipette tip in a spatiallyseparated manner, whereby the pipette tip is used anyway to perform aviscoelastic measurement or other diagnostic measurement. The resultingdegree of freedom to formulate the at least two separated parts (i.e.the at least two separated constituents) of the diagnostic compositionin either dry, essentially dry, liquid, or any other formulationcomprises a new and highly cost-effective approach. The presentinventors surprisingly arrived at the present invention based onperforming a combination of analyses of the mutual ingredientinteractions, performing stability studies in dependence on theformulation as dry, essentially dry or liquid, investigating stabilityand test performance studies to assess the possible negative impact ofthe employed containers on the test result in viscoelastic measurementswith body liquids like blood or blood plasma (cf. Examples) and, lastbut not least, understanding the commercial impact on providing areagent in either excess amount or not.

Preferably, each of the constituents (A) and (B) is independently fromeach other a liquid formulation, an essentially dry formulation, or adry formulation.

“Essentially dry” as used herein refers to a state, wherein the mixtureis essentially free from any liquid or moisture, in particular beingdepleted of water. Water or any other liquid, however, may be present asresidue in the mixture, but only to an extent, which does not negativelyinfluence the stability of the overall composition. In particular, ithas to be excluded that an interaction occurs between the differentconstituents, which negatively affects the stability. A remaining amountof liquid, preferably water, in the composition of up to 10% by weightmay be acceptable in an essentially dry formulation.

More preferably, constituents (A) and (B) are essentially dryformulations or dry formulations, and even more preferably constituents(A) and (B) are dry formulations. For example, it is preferred thateither (1) constituent (A) is a dry formulation and constituent (B) isan essentially dry formulation; or (2) constituent (A) is an essentiallydry formulation and constituent (B) is a dry formulation. Mostpreferably, however, both, constituents (A) and (B) are dryformulations.

Thereby, the stability of constituents (A) and (B) under roomtemperature conditions is enhanced. Nevertheless, constituents (A) and(B) may be filled into the pipette tip, e.g. during the manufacturingprocess of the pipette tip, in dry, essentially dry or in liquidformulation, for example by employing subsequent vacuum drying, heateddrying, lyophilization, or any other process suitable for drying theconstituents (A) and (B).

Preferably, in the pipette tip according to the present invention:

-   -   1) constituent (A) comprises component (i) but not        component (ii) and constituent (B) comprises component (ii) but        not component (i); or    -   2) constituent (A) comprises component (ii) but not        component (i) and constituent (B) comprises component (i) but        not component (ii).

Thereby, it is understood, that in situation 1), i.e. if component (i)is comprised by constituent (A) and component (ii) is comprised byconstituent (B), the constituent (A) does not comprise component (ii)and the constituent (B) does not comprise component (i). Accordingly, insituation 2), i.e. if component (ii) is comprised by constituent (A) andcomponent (i) is comprised by constituent (B), the constituent (A) doesnot comprise component (i) and the constituent (B) does not comprisecomponent (ii). Thus, each of the constituents (A) and (B) preferablycomprises either component (i) or component (ii).

According to the present invention component (i) of the diagnosticcomposition, i.e. the activator of coagulation, is preferably spatiallyseparated from component (ii) of the diagnostic composition, i.e. thecalcium salt. If these components remain spatially separated untilshortly before the viscoelastic analysis starts, a superior stability ofthe diagnostic composition (or the respective components) can beachieved and stability related problems can be avoided. Thus, theconstituent (A) of the diagnostic composition preferably compriseseither component (i) or component (ii), but not both, component (i) and(ii). Accordingly, the constituent (B) of the diagnostic compositionpreferably comprises the component selected from component (i) andcomponent (ii), which is not comprised by constituent (A), and also notboth, component (i) and (ii).

Although certain embodiments may be preferred, as described below, ingeneral either the activator of coagulation (component (i)) may becontained in constituent (A) and the calcium salt (component (ii)) maybe contained in constituent (B) or vice versa.

Diagnostic Composition and Constituents and Components Thereof

In the context of the present invention, the term “diagnosticcomposition” refers to a reagent composition (reagent mixture) forin-vitro diagnostic analysis, such as coagulation testing, in particularviscoelastic analysis, which is ready-to-use. In other words, inaddition to the diagnostic composition and the sample no further reagentis required to perform the diagnostic analysis. Moreover, dilution orthe like of the diagnostic composition is not necessary.

In the present invention, the constituents of the diagnosticcomposition, in particular the constituent (A) and the constituent (B),are spatially separated. As long as the constituents, in particular theconstituent (A) and the constituent (B), are spatially separated, theydo not yet form a diagnostic composition, however, they are able to forma diagnostic composition. The diagnostic composition is formed bybringing the constituents, e.g. constituent (A) and constituent (B),into contact with each other, preferably by mixing.

The sample may be brought into contact either (i) with the diagnosticcomposition, i.e. after contacting the constituents of the diagnosticcomposition with each other, or (ii) with a constituent, e.g. withconstituent (A) or constituent (B). In case (ii) the diagnosticcomposition is formed after contacting the sample with one of theconstituents, i.e. the sample is brought into contact with one of theconstituents, e.g. with constituent (A), and the other constituent, e.g.constituent (B), is contacted thereafter with a mixture of constituent(A) and the sample, thereby forming a mixture of the diagnosticcomposition and the sample.

According to the present invention, the pipette tip containsconstituents (A) and (B). When the sample is aspirated by the pipettetip the sample either contacts first constituent (A) or constituent (B),or both constituents at about the same time. Even if there are timedifferences between these two contacts, these temporal differences arerather negligible (typically less than 1 sec) and a mixture of thediagnostic composition and the sample is formed rather instantaneously.

As described above, a diagnostic composition comprises components, whichare described in more detail below. Since the constituents of thediagnostic composition are to form the diagnostic composition, theconstituents, in particular constituent (A) and constituent (B),comprise the components of the diagnostic composition. Thereby, one ormore components of the diagnostic composition may be comprised byconstituent (A), one or more components of the diagnostic compositionmay be comprised by constituent (B), and one or more components of thediagnostic composition may be comprised by both, constituents (A) and(B).

Thus, it is understood, that a component “comprised by the diagnosticcomposition” is a component, which is comprised by one or both of theconstituents, i.e. by the constituents (A) and/or (B). In other words,if the diagnostic composition comprises a certain component, thiscomponent is usually comprised by constituent (A) (and not byconstituent (B)) or by constituent (B) (and not by constituent (A)) orby both, constituent (A) and (B).

In a preferred embodiment, the diagnostic composition may be forviscoelastic analysis. Thereby, the constituents (A) and/or (B),preferably comprise(s) (i) an activator of coagulation (e.g., anactivator of either the intrinsic or the extrinsic pathway), (ii) acalcium salt, and (iii) optionally one or more further inhibitors, e.g.fibrinolysis inhibitors, platelet inhibitors, heparin inhibitors and/or(iv) optionally one or more further specific factors or co-factors ofthe coagulation cascade.

In another preferred embodiment, the diagnostic composition may be forcoagulation diagnostics in blood or blood plasma other than viscoelasticanalysis, such as ‘Prothrombin Time’ (PT), ‘Activated PartialThromboplastin Time’ (APTT), ‘Activated Clotting Time’ (ACT), or‘Prothrombinase Induced Clotting Time’ (PICT). Thereby, the constituentsA and B are preferably exactly the same as described herein forviscoelastic analysis or the activator may be different. Thus, preferredcoagulation activators include FXa and Russel's Viper Venom—Factor Vactivating component (RVV-V). Moreover, in this embodiment, it may notbe necessary to provide a calcium salt in one of the constituents (A) or(B), but to separate other components by dividing them into constituent(A) and constituent (B). For example, in PICT diagnostics the enzyme FXashould be separated from the protein component RVV-V, because theactivity of these substances is compromised when they are applied incombination.

Preferably, the activator of coagulation is an extrinsic activatorand/or an intrinsic activator, i.e. an activator of the extrinsicpathway (the Tissue Factor pathway) or of the intrinsic pathway (theContact Activation pathway).

Thereby, it is preferred that:

-   -   component (i) is an extrinsic activator of coagulation and        component (ii) is a calcium salt; or    -   component (i) is an intrinsic activator of coagulation and        component (ii) is a calcium salt.

The extrinsic activator of coagulation maybe an activator of theExtrinsic Prothrombin Activation Pathway (extrinsic pathway), inparticular a Tissue factor (TF, also referred to as platelet tissuefactor, factor III, thromboplastin, or CD142). Preferably, the TF isselected from lipidated TF or recombinant TF (rTF).

The intrinsic activator of coagulation maybe any activating factor ofthe Contact Activation pathway (intrinsic pathway), e.g., celite,ellagic acid, sulfatit, kaolin, silica, or RNA. Preferably, theintrinsic activator of coagulation is selected from celite, ellagicacid, sulfatit, kaolin, silica, RNA, and mixtures thereof.

The pipette tip according to the present invention as described hereinmay contain a calcium salt, preferably as component (ii). The calciumsalt is added for re-calcification of the sample. Blood samples can beprevented from clotting by several different anticoagulatory substanceslike heparin, EDTA, citrate. Typically functional tests are performedwith blood anticoagulated with citrate. Citrate moderately complexescalcium of the blood sample. Calcium is necessary for the coagulationprocess, it is involved in complex formation and is a co-factor for mostof the coagulation factors (e.g., FI, FII, FV, FVII, FVIII, FIX, FX,FXI, FXIII, TF). Therefore, recalcification of the sample is necessaryto ensure correct coagulation in the sample, if the sample was forexample citrated during blood withdrawal (by using a blood tubecontaining citrate). Preferably, the calcium salt is calcium chlorideand/or calcium lactate and/or calcium gluconate. More preferably, thecalcium salt is CaCl₂.

Preferably, the calcium salt, in particular CaCl₂, is present in anamount of about 1-100 μmol/ml of sample (test liquid), more preferablyin an amount of about 3-30 μmol/ml of sample (test liquid). As mentionedabove, the amount of the calcium salt, in particular CaCl₂, must besufficient to ensure recalcification of the sample, in particular of theblood sample, if the sample was decalcified before. It turned out thatan amount of from 3-30 μmol/ml is particularly optimal to achieve thisrequirement. In order to determine the required amount of the calciumsalt, in particular CaCl₂, to be contained in the diagnosticcomposition, i.e. the constituents (A) and/or (B), even more precisely,the exact volume of the sample to be collected from the patient has tobe known as well as the amount of decalcifying reagent employed.

The diagnostic composition, i.e. the constituents (A) and/or (B), mayoptionally comprise further components. Preferably, the diagnosticcomposition, i.e. the constituents (A) and/or (B), further comprises oneor more components selected from the group consisting of: a furtheractivator of coagulation (i.e. an activator of coagulation as describedherein, which is different from the activator of coagulation comprisedas component (i); also referred to as “coagulating activating factor); acoagulation inhibitor (i.e., a substance that stops, reduces, or atleast modifies the function of a certain components of the coagulationand/or clot lysis cascade); and an active-component inhibitor (i.e., asubstance the stops, reduces, or at least modifies the function of acomponent active in coagulation, e.g. a coagulation inhibitor).Preferably, the coagulation activating factor is selected from the groupconsisting of FI, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, and TF.Preferably, the coagulation inhibitor is selected from the groupconsisting of tissue factor pathway inhibitor, antithrombin I-IV, oractivated protein C.

Preferably, the active-component inhibitor is selected from the groupconsisting of one or more platelet inhibitors (i.e., substances thatstop, reduce, or at least modify the function of thrombocytes), one ormore fibrinolysis inhibitors (i.e., substances that stop, reduce, or atleast modify the function of clot lysis), and/or one or more heparininhibitors. Preferably, the platelet inhibitor is a cytoskeletoninhibitor, preferably Cytochalasin D, or a GPIIb/IIIa antagonist,preferably Abciximab. Preferably, the fibrinolysis inhibitor is selectedfrom the group consisting of aprotinin, tranexamic acid, eaca,thrombin-activated fibrinolysis inhibitor, plasminogen activationinhibitor 1/2, α2-antiplasmin, and α2-macroglobulin. Preferably, theheparin inhibitor is selected from heparinase, protamine orprotamine-related peptides and their derivatives, or other cationicpolymers, for example hexadimethrine bromide (polybrene). The heparininhibitor is in particular useful to detect the presence of heparin inthe sample and, thus, the amount of heparin inhibitor (e.g., heparinase)is a sufficient to detect the presence of heparin in the sample.

Those inhibitors may be used and combined depending on the precisediagnostic demands, for example, the platelet inhibitor may be acytoskeleton inhibitor or a GPIIb/IIIa antagonist. The fibrinolysisinhibitor can be selected, for example, from aprotinine, tranexamicacid, or eaca; the heparin inhibitor might be selected, for example,from heparinase, protamine or protamine-related peptides; and thecoagulation factor can be selected, for example, from one or morecoagulation factors or activated coagulation factors preferably FXa orFVa or activated protein C or FVIIa. However, it is noted that this isonly a preferred selection and further inhibitors can be used ifrequired.

Preferably, the diagnostic composition, i.e. the constituents (A) and/or(B), may also contain one or more stabilizers, wherein the stabilizer ispreferably albumin or gelatine. Such stabilizers are typically used forthe stabilization of the reagents between the time of production and theanalysis. Preferably, in the pipette tip according to the presentinvention, a protein stabilizer, for example albumin or gelatin, iscomprised by constituent (A) but is not comprised by constituent (B); ora protein stabilizer, for example albumin or gelatin, is comprised byconstituent (B) but is not comprised by constituent (A).

Alternatively, it is also preferred that the diagnostic composition,i.e. the constituents (A) and/or (B), do(es) not contain albumin, morepreferably the diagnostic composition, i.e. the constituents (A) and/or(B), do(es) not contain albumin or gelatin, and even more preferably thediagnostic composition, i.e. the constituents (A) and/or (B), do(es) notcontain any stabilizer. Most preferably, the pipette tip, and inparticular the constituents (A) and (B), do not contain proteinstabilizers like albumin. Such stabilizers may interfere withviscoelastic measurements.

Preferably, the diagnostic composition, i.e. the constituents (A) and/or(B), may also contain one or more phospholipids. Phospholipids may beadded since several complexes in the coagulation cascade arephospholipid-dependent. Preferably, the phospholipids may be acomposition of different phospholipids like for examplephosphatidylserine, phosphatidylethanolamine andphosphatidylethanolcholine. Preferably, mixtures of differentphospholipids as extracted from biological samples (e.g., rabbit brain)may be used.

Preferably, the phospholipid and/or the heparin inhibitor, preferablyheparinase and/or hexadimethrine bromide (polybrene), more preferablyhexadimethrine bromide (polybrene), are comprised by constituent (A) andarranged in compartment (a).

Depending on the diagnostic aim, the above described components can beused either alone or in combination: For example, a measurement withonly an intrinsic activator in the sample can be combined with ameasurement with an intrinsic activator and a sufficient amount ofheparin inhibitor (e.g., heparinase) in the sample to detect thepresence of heparin in the test liquid; a combination of extrinsicactivator and platelet inhibitor (e.g., Cytochalasin D) in the samplecan be applied to determine the activity of fibrinogen without plateletcontribution in the sample.

Preferably, the diagnostic composition, i.e. the constituents (A) and/or(B), comprise or consist of a combinations of components selected fromthe following combinations of components:

-   -   extrinsic activation: Combination of extrinsic activator and        CaCl₂ and, optionally, stabilizer(s);    -   intrinsic activation: Combination of intrinsic activator and        CaCl₂ and, optionally, stabilizer(s);    -   extrinsic activation insensitive for heparin: Combination of        extrinsic activator, heparin inhibitor, CaCl₂ and, optionally,        stabilizer(s);    -   intrinsic activation insensitive for heparin: Combination of        intrinsic activator, heparin inhibitor, CaCl₂ and, optionally,        stabilizer(s);    -   extrinsic activation without platelet activation: Combination of        extrinsic activator, platelet inhibitor and CaCl₂ and,        optionally, stabilizer(s);    -   extrinsic activation without platelet activation, insensitive        for heparin: Combination of extrinsic activator, platelet        inhibitor, heparin inhibitor, CaCl₂ and, optionally,        stabilizer(s);    -   extrinsic activation without platelet activation, insensitive        for heparin: Combination of extrinsic activator, platelet        inhibitor, heparin inhibitor, CaCl₂ and, optionally,        stabilizer(s);    -   intrinsic activation without platelet activation: Combination of        intrinsic activator, platelet inhibitor, CaCl₂ and, optionally,        stabilizer(s);    -   intrinsic activation without platelet activation, insensitive        for heparin: Combination of intrinsic activator, platelet        inhibitor, heparin inhibitor, CaCl₂ and, optionally,        stabilizer(s);    -   extrinsic activation with inhibition of fibrinolysis:        Combination of extrinsic activator, fibrinolysis inhibitor and        CaCl₂ and, optionally, stabilizer(s);    -   extrinsic activation with inhibition of fibrinolysis,        insensitive for heparin: Combination of extrinsic activator,        fibrinolysis inhibitor, heparin inhibitor, CaCl₂ and,        optionally, stabilizer(s);    -   intrinsic activation with inhibition of fibrinolysis:        Combination of intrinsic activator, fibrinolysis inhibitor and        CaCl₂ and, optionally, stabilizer(s);    -   intrinsic activation with inhibition of fibrinolysis,        insensitive for heparin: Combination of intrinsic activator,        fibrinolysis inhibitor, heparin inhibitor, CaCl₂ and,        optionally, stabilizer(s);    -   extrinsic activation with additional coagulation factor:        Combination of extrinsic activator, one additional coagulation        factor and CaCl₂ and, optionally, stabilizer(s);    -   extrinsic activation with additional coagulation factor,        insensitive for heparin: Combination of extrinsic activator, one        additional coagulation factor, heparin inhibitor, CaCl₂ and,        optionally, stabilizer(s);    -   intrinsic activation with additional coagulation factor:        Combination of intrinsic activator, one additional coagulation        factor and CaCl₂ and, optionally, stabilizer(s);    -   intrinsic activation with additional coagulation factor,        insensitive for heparin: Combination of intrinsic activator, one        additional coagulation factor, heparin inhibitor, CaCl₂ and,        optionally, stabilizer(s).

In a first preferred embodiment, constituent (A) comprises at least anextrinsic activator of coagulation, which is preferably selected fromTissueFactor (TF), lipidated TF or recombinant TF or any mixturesthereof in dry, essentially dry, or liquid formulation or in any otherformulation that allows for dissolution of the TF within 30 s afteraspiration of the sample liquid. In the same preferred embodiment,constituent (B) comprises a calcium salt. Preferably, constituents (A)and (B) do not comprise a platelet inhibitor and/or a fibrinolysisinhibitor. This embodiment is referred to as ‘EX-tip’ in the followingand could be used to perform a viscoelastic measurement of theextrinsically activated coagulation pathway.

In a second preferred embodiment, constituent (A) and constituent (B)comprise the components as described for the first preferred embodiment.In addition, either constituent (A) or constituent (B) as described forthe first preferred embodiment contains additionally one or moreplatelet inhibitors, preferably selected from GPIIb/IIIa antagonists,preferably Abciximab, and/or cytoskeleton inhibitors, preferablyCytochalasin D, in dry, essentially dry, or liquid formulation, or inany other formulation that allows for dissolution of the reagentcomposition within 30 s after aspiration of the sample liquid. Thisembodiment is referred to as ‘FIB-tip’ in the following.

In a third preferred embodiment, constituent (A) and constituent (B)comprise the components as described for the first preferred embodiment.In addition, either constituent (A) or constituent (B) from the firstpreferred embodiment contains additionally one or more fibrinolysisinhibitors, preferably aprotinine, tranexamic acid or eaca, in dry,essentially dry, or liquid formulation, or in any other formulation thatallows for dissolution of the reagent composition within 30 s afteraspiration of the sample liquid. This embodiment is referred to as‘AP-tip’ in the following.

It is also preferred that constituent (A) or (B) according to the threepreviously described preferred embodiments ‘EX-tip’, ‘FIB-tip’ and‘AP-tip’ contains in addition one or more heparin inhibitors, preferablyprotamine or protamine derivatives, whereby preferred protaminederivatives include protamine sulfate and protamine hydrochloride, orother protamine-like peptides and their derivatives, or other cationicpolymers, preferably hexadimethrine bromide (polybrene), that have thepotential to neutralize the anti-coagulating effect(s) of heparine orheparine-like substances in a blood sample by charge interaction. Theresulting reagent composition is preferably a dry, essentially dry or aliquid formulation, or any other formulation that allows for dissolutionof the reagent composition within 30 s after aspiration of the sampleliquid. Corresponding tips are referred to as ‘EX-tip HI’, ‘FIB-tip HI’,and ‘AP-tip HI’ in the following.

In a fourth preferred embodiment, constituent (B) comprises at least anintrinsic activator of coagulation, which is preferably selected fromcelite, ellagic acid, sulfatit, kaolin, silica, RNA, or any mixturesthereof in dry, essentially dry, or liquid formulation, or in any otherformulation that allows for dissolution of the activator within 30 safter aspiration of the sample liquid. In this embodiment, theconstituent (A) comprises a calcium salt and the embodiment is referredto as ‘IN-tip’ in the following.

In a fifth preferred embodiment, constituent (A) and constituent (B)comprise the components as described for the fourth preferredembodiment. In addition, either constituent (A) or (B) of the ‘IN-tip’embodiment comprises additionally one or more heparin inhibitors,preferably heparinase, protamine, or protamine-related peptides, in dry,essentially dry or liquid formulation, or in any other formulation thatallows for dissolution of the activator within 30 s after aspiration ofthe sample liquid. This embodiment is referred to as ‘HEP-tip’ in thefollowing.

It is also preferred that the calcium salt in all embodiments mentionedabove is selected from CaCl₂, Calcium-Lactate, Calcium-Gluconate, or anymixtures thereof in dry, essentially dry, or liquid formulation, or inany other formulation that allows for dissolution of the activatorwithin 30 s after aspiration of the sample liquid.

If the diagnostic composition comprises more than two components to beseparated in compartments (a) and (b), the additional components mayalso be placed in additional compartments (c), (d), etc. within the tip.Thus, the pipette tip may contain one or more compartments, for example1, 2, 3, 4, or 5 compartments. Preferably, the more than twocomponent(s) are combined, e.g. mixed, with constituent(s) (A) and/or(B), and, thus, compartments (a) and (b) are sufficient. However, it isalso preferred that additional components, which are not comprised byconstituent(s) (A) and/or (B) may be placed in additional compartmentsother than compartments (a) and (b).

If additional components of the diagnostic composition are mixed witheither constituent (A) or (B), they must be in the same physicalcondition (i.e., liquid, dry, essentially dry, etc.). If they are placedin an additional compartment, they can have another physical condition.For example, one component in a first compartment is a dry formulation,while another component in a second compartment is a liquid formulation,and a third component is essentially dry formulation. In another exampletwo components are mixed as liquids and one component may be dried in aseparate compartment, or, in another example, two components are a drymixture and one component may be a dried liquid in a separatecompartment.

Preferred embodiments of a pipette tip containing constituents (A) and(B) in compartments (a) and (b), respectively, are shown in FIGS. 3 a-3g . A first preferred embodiment, shown in FIG. 3 a ), is a pipette tip(11 a), which has a conventional (merely conical) tip shape with openlower end (12 a) and open upper end (13 a) fitting to the pipettedimensions. Compartment (b) containing constituent (B) is represented bya porous insert (14 a). Compartment (a) containing constituent (A) isrepresented by the lowest cross-wise section of the tip (below theporous insert (14 a)) having an inner tip surface area of 1-1000 mm², onwhich a circumferential reagent layer (15 a) of constituent (A) isdeposited.

Another preferred embodiment, shown in FIG. 3 b ), is a pipette tip (11b) having a modified tip shape with open lower end (12 b) and open upperend (13 b) fitting to the pipette dimensions. Compartment (b) containingconstituent (B) is represented by a porous insert (14 b), wherein theporous insert (14 b) has a cylindrical shape. Compartment (a) containingconstituent (A) is represented by the lowest cross-wise section of thetip (below the porous insert (14 a)) having an inner tip surface area of1-1000 mm², on which a spot-like (not circumferential) reagent layer (15a) of constituent (A) is deposited.

Another preferred embodiment, shown in FIG. 3 c ), is a pipette tip (11c) having a modified tip shape with open lower end (12 c) and open upperend (13 c) fitting to the pipette dimensions. Pipette tip (11 c) has twoporous inserts (14 c and 14 c′) forming compartment (a) comprisingconstituent (A) (14 c) and compartment (b) comprising constituent (B)(14 c′), the two porous inserts positioned adjacently to each other,wherein each of the porous inserts (14 c, 14 c′) has a cylindrical shapewith preferably the same diameter.

Another preferred embodiment, shown in FIG. 3 d ), is a pipette tip (11d), which has a conventional (merely conical) tip shape with open lowerend (12 d) and open upper end (13 d) fitting to the pipette dimensions.Compartment (b) containing constituent (B) is represented by a porousinsert (14 d). Compartment (a) containing constituent (A) is representedby the cross-wise section of the tip just above the porous insert (14 d)having an inner tip surface area of 1-1000 mm², on which acircumferential reagent layer (15 d) of constituent (A) is deposited.

Another preferred embodiment, shown in FIG. 3 e ), is a pipette tip (11e), which has a conventional (merely conical) tip shape with open lowerend (12 e) and open upper end (13 e) fitting to the pipette dimensions.Compartment (a) containing constituent (A) is represented by the lowestcross-wise section of the tip having an inner tip surface area of 1-1000mm², on which a circumferential reagent layer (15 e) of constituent (A)is deposited. Compartment (b) containing constituent (B) is representedby the cross-wise section of the tip just above compartment (a) havingan inner tip surface area of 1-1000 mm², on which a circumferentialreagent layer (15 e′) of constituent (B) is deposited.

Another preferred embodiment, shown in FIG. 3 f ), is a pipette tip (11f), which has a conventional (merely conical) tip shape with open lowerend (12 f) and open upper end (13 f) fitting to the pipette dimensions.Compartment (a) containing constituent (A) is represented by the rightlongitudinal section of the tip, on which a spot-like (notcircumferential) reagent layer (15 f) of constituent (A) is deposited.Compartment (b) containing constituent (B) is represented by the leftlongitudinal section of the tip, on which a spot-like (notcircumferential) reagent layer (15 f′) of constituent (B) is deposited.

In further preferred embodiments, an additional compartment (c) may bepresent, which may either be represented by an additional porous insertor by an additional tip section containing a reagent layer. Such acompartment (c) may be located in the pipette tip (i) above both,compartments (a) and (b); (ii) below both, compartments (a) and (b); or(iii) between compartment (a) and compartment (b).

Taken together, one of the several advantages of the pipette tipaccording to the present invention is that the constituent (A) containedin compartment (a) and the constituent (B) contained in compartment (b)can be optimized with regard to the long-term stability of thediagnostic composition. For example, if the TF has less stability whenmixed with hexadimethrine bromide (polybrene), the TF can be placed inone compartment and the hexadimethrine bromide (polybrene) can be placed(optionally together with the calcium salt) in versa separatedcompartment. For example, if the TF has also less stability when mixedwith Cytochalasin D, Cytochalasin D can also be placed in thecompartment where the polybrene is placed. But, for example, ifCytochalasin D has less stability when mixed with hexadimethrine bromide(polybrene) in liquid phase, it can be eventually placed together withthe TF in one compartment.

Thus, the pipette tip according to the present invention provides aconsiderable variety of options to increase reagent stability to therequired level. This is achieved without the need for new automationtechnology for reagent filling or reagent handling and without addingnew substances to the reagent composition.

Furthermore, it is preferable that the constituent (A) and/or theconstituent (B) described herein additionally comprise a coagulationfactor, preferably selected from FI, FII, FV, FVII, FVIII, FIX, FX, FXI,and FXIII or a coagulation inhibitor, preferably selected from TFPI,ATIII and APC.

Method and Use

In a second aspect the present invention provides a method of performinga diagnostic test on a sample, preferably a body fluid or blood sample,comprising the following steps:

-   -   (1) providing a pipette tip according to the present invention        as described above;    -   (2) aspirating the sample into the pipette tip, thereby mixing,        preferably dissolving, the constituent (A), e.g. contained in        compartment (a), and the constituent (B), e.g. contained in        compartment (b), of said pipette tip in the sample and obtaining        a mixture, preferably a solution, of sample and constituents (A)        and (B), wherein said mixture of constituent (A) and (B) form a        diagnostic composition that is required to perform a diagnostic        test;    -   (3) optionally, transferring the mixture of said sample and said        diagnostic composition into a measurement container, such as a        cuvette, suitable for performing said diagnostic test;    -   (4) optionally, putting the measurement container, such as the        cuvette, into an apparatus suitable for performing said        diagnostic test; and    -   (5) performing the diagnostic test of said mixture, optionally        in the measurement container, such as a cuvette.

The sample is preferably a blood sample or a sample comprising afraction of blood (e.g., isolated plasma or platelets), more preferablymammalian, in particular human blood or blood components or a fractionof blood. For example, the sample is whole blood or blood plasma, inparticular human whole blood or human blood plasma. The sample maycontain additional components added ex-vivo to the sample, for exampleacids, bases or buffers for modification, correction or stabilization ofpH levels; and/or salts for modification, correction or stabilization ofion levels; and/or diluents to adjust the concentration of samplecomponents to a required level like water, protein solutions, colloidsolutions or and/or modifiers of the physical properties of the sample(e.g., oils or organic solvents to increase or decrease the sampleviscosity; or surfactants to increase/decrease foam formation, etc.).

The diagnostic test performed in the method according to the presentinvention is preferably assessing the coagulation status of the sampleand is, thus, a “coagulation test”. Coagulation tests (also referred toas “blood clotting tests”) are the tests used for diagnostics of thehemostasis system. Preferably, the coagulation test is a globalcoagulation test or a local coagulation test. Global tests characterizethe results of work of the whole clotting cascade. They suit to diagnosethe general state of the blood coagulation system and the intensity ofpathologies, and to simultaneously record all attendant influences.Global methods play the key role at the first stage of diagnostics: theyprovide an integral picture of alterations within the coagulation systemand allow predicting a tendency to hyper- or hypo-coagulation ingeneral. Local tests characterize the results of work of the separatecomponents of the blood coagulation system cascade, as well as of theseparate coagulation factors. They are essential for the possibility tospecify the pathology localization within the accuracy of coagulationfactor. Preferred examples of a global coagulation tests includethromboelastography, thrombin generation test (thrombin potential,endogenous thrombin potential) and thrombodynamics test. Preferredexamples of a local coagulation tests include Partial thromboplastintime (PTT or APTT: activated partial thromboplastin time), Prothrombintime test (or prothrombin test, INR, PT) and other highly specializedmethods to reveal the alteration in concentration of separate factors.

Preferably, the coagulation test measures:

-   (i) the time delay between coagulation activation and initial    clotting (e.g., Prothrombin Time (PT), International Normalized    Ratio (INR), Partial ThromboPlastin Time (PTT), activated Partial    ThromboPlastin Time (aPTT), etc.); and/or-   (ii) the platelet aggregation activity (e.g., by optical    aggregometry or impedance aggregometry); and/or-   (iii) the clot strength (e.g., by viscoelastic methods like    thromboelastography or thromboelastometry); and/or-   (iv) the clot lysis activity (e.g., by D-dimer level detection or    clot strength decrease in viscoelastic methods).

In a coagulation test, the sample is preferably a blood sample or afraction of a blood sample as described above.

Most preferably, the diagnostic test is a viscoelastic analysis.

Accordingly, it is preferred that the diagnostic test in step (5)comprises the determination of the clotting time, the clot formationtime, the firmness of the clot over time, fibrinolysis activity(obtained as percentage of firmness reduction in relation to the maximumclot firmness), and/or any combination thereof.

The apparatus suitable for performing a diagnostic test, such as aviscoelastic analysis, is preferably a device as described in U.S. Pat.No. 5,777,215 A or in U.S. Pat. No. 6,537,819 B2. Another preferredexample of an apparatus suitable for performing a viscoelastic analysisis schematically shown in FIG. 2 . In more general, it is preferred thatthe apparatus suitable for performing a diagnostic test is acoagulometer. A coagulometer is a medical laboratory analyzer used fortesting of the hemostasis system, in particular in coagulation tests.Modern coagulometers realize different methods of activation andobservation of development of blood clots in blood or in blood plasma.

In more general, as used herein a viscoelastic analysis (also referredto as viscoelastic measurement) refers to a (viscoelastic) analysis of asample, in particular a blood sample or a sample of blood elements, e.g.plasma or cells, in order to determine its coagulation characteristics,wherein such a viscoelastic analysis in the broadest sense is themeasurement of a relative movement of a measurement container, such as acuvette, containing a blood sample relative to a pin. In particular, ina typical viscoelastic analysis a clot is formed between measurementcontainer and pin and thereafter the clot itself is stretched by themovement of the pin relative to the container. The detection of thecharacteristic parameters of the clot is based on the mechanicalcoupling of container and pin by the clot. In particular, a viscoelasticmeasurement provides information about several distinct parameters, forexample the time between coagulation activation and clot initiation(clotting time CT), the dynamics of clot formation (clot formation timeCFT), the firmness of the clot (amplitudes A5-A30 and maximum clotfirmness MCF), and/or the extent of fibrinolysis (maximum lysis ML).Thus, the viscoelastic analysis preferably comprises the determinationof the clotting time, the clot formation time, and/or the firmness ofthe clot over time including fibrinolytic effects. An exemplary diagramshowing a typical viscoelastic analysis (also referred to asviscoelastic measurement) and the meaning of the parameters mentionedabove is shown in FIG. 1 . The clotting time CT is the initial lag timeuntil the firmness starts to build up. The amplitude values A5-A30 arethe firmness values 5-30 minutes after CT. The maximum lysis is thepercentage decrease of firmness after the maximum value (MCF) wasreached.

The positioning of the measurement container into an apparatus suitablefor performing the diagnostic test (step (4)) is optional, since it mayalso occur at any time before. For example, all pipetting can also beperformed while the measurement container is in the apparatus, if theapparatus provides enough access to the upper open end of themeasurement container while placed in measurement position.

Preferably, the method of performing a diagnostic test, such as aviscoelastic analysis, on a sample according to the present inventionfurther comprises a step (2-a) that follows step (2) and precedes step(3), wherein the tip is kept on the pipette for 1-30 s, preferably from1-5 s. Thereby, a better or even complete reagent dissolution isallowed. Accordingly, in step (2-a) a short time delay of e.g. 1-30 sallows for complete dissolution of constituents (A) and (B) of thediagnostic composition within the sample. Additionally, the samplewithin the tip could be moved gently up and down within the pipette tipduring step (2-a) by using the aspiration/dispension functionality ofthe pipette, which is preferably realized in an automated sequence thatdoes not require any user activity.

Preferably, each of steps (2) and (3) of the method of the presentinvention takes less than 30 sec, more preferably each of steps (2) and(3) takes from 2 to 10 s. Thereafter, the mixture of the sample and thediagnostic composition (optionally in the measurement container) ispreferably quickly transferred to the measuring apparatus, morepreferably in less than 30 s.

It is also preferred in the method according to the present inventionthat the method further comprises a step (3-a) following step (3) andpreceding step (4), wherein in step (3-a) the mixture is at leastpartially re-aspired into the pipette tip and subsequently releasedagain into the measurement container. This step may be repeated one ormore, e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Thereby a better mixingof the sample is achieved and a better dissolution of constituents (A)and (B) is allowed.

No change of pipette tip is necessary when preparing one test. Thisshows the direct benefit of the present invention for the person who isperforming such tests regarding the ease of use.

In a further aspect, the present invention also provides the use of apipette tip as described above in a coagulation test as described above.Preferably, the pipette tip as described above is used in viscoelasticanalysis as described above.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will begiven. The figures are intended to illustrate the present invention inmore detail. However, they are not intended to limit the subject matterof the invention in any way.

FIG. 1 is an exemplary diagram showing a typical viscoelasticmeasurement and corresponding curve parameters: clotting time CT is thelag time between activation of the sample and the time when a firmnessvalue of 2 mm is reached; clot formation time CFT is the time thatpasses between the firmness values of 2 mm and 20 mm; alpha is the anglethat is formed between the tangential of the firmness curve and thex-axis; maximum clot firmness MCF is the maximum firmness value of thecurve; maximum lysis is the percentage decrease of firmness after MCFhas been reached.

FIG. 2 shows an illustration of an apparatus for viscoelastic testing:After the formation of the clot between cup 1 (cuvette) and pin 2, theclot itself is stretched by the movement of the pin 2 relative to thecup 1. The detection of the characteristic parameters of the clot isbased on the mechanical coupling of cup 1 and pin 2 by the clot. This isonly possible if the clot adheres on the surfaces of both cup 1 and pin2. Thus, a firm adhesion to the surfaces of both cup 1 and pin 2 istypically required for the viscoelastic analysis. During a viscoelasticmeasurement, the pin is fixed to the axis 4 and gently and slowlyrotated in the cup via the spring 7. The axis 4 itself is fixed to thebase plate 5 with the ball bearing 6. The movement of the pin ismeasured optically by illuminating the mirror 9 (fixed to the axis 4)with the light source 8 and detecting the reflected signal at thespatially resolving photo detector 10.

FIG. 3 shows schematic cross-sectional views of six preferredembodiments of a pipetting tip (11) containing constituents (A) and (B):

-   -   a) longitudinal cross-section through a conventional pipette tip        (11 a) with open lower end (12 a), open upper end (13 a) fitting        to the pipette dimensions, porous insert (14 a) and reagent        layer (15 a) below the porous insert (14 a);    -   b) longitudinal cross-section through a modified pipette tip (11        b) with open lower end (12 b), open upper end (13 b) fitting to        the pipette dimensions, porous insert (14 b) and reagent layer        (15 b) below the porous insert (14 b);    -   c) longitudinal cross-section through a modified pipette tip        shape (11 c) with open lower end (12 c), open upper end (13 c)        fitting to the pipette dimensions, and two porous inserts (14 c,        14 c′);    -   d) longitudinal cross-section through a conventional pipette tip        (11 d) with open lower end (12 d), open upper end (13 d) fitting        to the pipette dimensions, porous insert (14 d) and reagent        layer (15 d) above the porous insert (14 d);    -   e) longitudinal cross-section through a conventional pipette tip        (11 e) with open lower end (12 e), open upper end (13 e) fitting        to the pipette dimensions, and two circumferential reagent        layers (15 e, 15 e′) located on top of each other; and    -   f) longitudinal cross-section through a conventional pipette tip        (11 f) with open lower end (12 f), open upper end (13 f) fitting        to the pipette dimensions, and two spot-like reagent layers (15        f, 15 f) located in juxtaposition.

LIST OF REFERENCE SIGNS

-   1, 1 a, 1 b, 1 c measurement cup-   2, 2 a, 2 b, 2 c pin-   3 sample-   4 axis-   5 base plate-   6 ball bearing-   7 spring-   8 light source-   8 mirror-   10 detector-   11, 11 a, 11 b, 11 c, 11 d, 11 e, 11 f pipette tip-   12 a, 12 b, 12 c, 12 d, 12 e, 12 f open lower end of the pipette tip-   13 a, 13 b, 13 c, 13 d, 13 e, 13 f open upper end of the pipette tip-   14 a, 14 b, 14 c, 14 c′, 14 d porous insert-   15 a, 15 b, 15 d, 15 e, 15 e′, 15 f, 15 f′ reagent layer

EXAMPLES

In the following, particular examples illustrating various embodimentsand aspects of the invention are presented. However, the presentinvention shall not to be limited in scope by the specific embodimentsdescribed herein. The following preparations and examples are given toenable those skilled in the art to more clearly understand and topractice the present invention. The present invention, however, is notlimited in scope by the exemplified embodiments, which are intended asillustrations of single aspects of the invention only, and methods whichare functionally equivalent are within the scope of the invention.Indeed, various modifications of the invention in addition to thosedescribed herein will become readily apparent to those skilled in theart from the foregoing description, accompanying figures and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1 Effect of TF/Phospholipids Deposited Either in Compartment (a)or in Compartment (b) of the Pipette Tip on Clotting Time

To investigate the effect of the extrinsic activator of coagulation onthe clotting time when TF and phospholipids are deposited in dry formeither in compartment (a) or in compartment (b), viscoelasticmeasurements of human plasma samples (10 donors mixed) were performedwith a ROTEG® 05 device (Pentapharm GmbH, Munich, Germany). In thepipette tip used herein, compartment (b) was formed by a cylindricalporous insert made of polyether foam (RG 130 grey, Hildebrandt andRichter & Co. GmbH, Germany), the porous insert having a cylindricalshape of 5 mm height and 4 mm diameter. The porous insert was located inthe lower half of the pipette tip. Compartment (a) of the pipette tipwas provided by a crosswise section of the pipette tip, wherein aspot-like reagent layer of approximately 1.5 mm diameter (correspondingto about 4 it liquid reagent before drying) was deposited.

Frozen plasma samples where freshly thawed and heated to 37° C. justbefore measurement. The source of TF/phospholipids was Innovin® (SiemensAG, Germany) and the source of CaCl₂ was Calcium Chloride Dihydrate(Sigma-Aldrich Chemie GmbH, Germany). Pipetting of TF was performed withTop-Line® 1 ml tips (AHN Biotechnologie GmbH, Germany) on a manual 1 mlpipettor (Brand, Germany). Liquid CaCl₂ was placed in the measurementcuvette just before TF pipetting was performed. The TF compositioncontained 15 ul of Innovin® standard stock solution together with 4%sucrose and was dried for 2 days in a desiccator filled with 100 gmolecular sieve (4 Angstroem) after placing either in compartment (a) orin compartment (b). For the control experiment, the same sample wasmeasured by using the standard liquid reagent provided for the ROTEG® 05system (TEM Innovations GmbH, Germany).

Results are shown in Table 1 below.

TABLE 1 Clotting times (CT) obtained after placing the same amounts ofTF in the pipette tip in either dry or wet form either in compartment(a) or in compartment (b). Each value was calculated as average of 4measurements with human plasma. Mixed storage of TF and CaCl₂ impairsthe clotting time CT severely (correction impossible), while mixing TFwith 4% sucrose can compensate for degradation during storage and/ordissolution delays of pure TF. CT of dry CT of dry CT of wet CT of wetTF in TF in TF in TF in com- com- com- com- CT partment partmentpartment partment Activator control (a) (b) (a) (b) Tissue factor/ 57sec 61 sec 239 sec 55 sec 168 sec phospholipids

Example 2 Effect of TF/Phospholipids Deposited in the Pipette Tip Aloneor in Combination with CaCl₂ on Clotting Time

To investigate the effect of the extrinsic activator of coagulation TFand phospholipids deposited in dry form in compartment (a) of thepipette tip either alone or in combination with CaCl₂ on the clottingtime, viscoelastic measurements of human plasma samples (10 donorsmixed) were performed with a ROTEG® 05 device (Pentapharm GmbH, Munich,Germany) according to the procedures described in Example 1. Compartment(a) of the pipette tip was provided by a crosswise section in the lowerthird of the pipette tip, wherein a spot-like reagent layer of about 2mm diameter, corresponding to about 5 μl liquid reagent before drying,was deposited.

Results are shown in Table 2 below.

TABLE 2 Clotting times (CT) obtained after drying the same amounts of TFsolution in compartment (a) of the pipette tip and storing for one weekat room temperature. For the pure TF sample, the same amount of CaCl₂ asin the mixed sample was added just before the measurement (each valuewas calculated as average of 4 measurements with human plasma). Mixedstorage of TF and CaCl₂ impairs the clotting time CT severely(correction impossible), while increasing the amount of TF by a factorof 4 and adding 2% sucrose can compensate for degradation of the pure TFsample during storage and/or dissolution delays. CT of TF/CaCl₂ CT of CTof pure TF Activator CT control mixture pure TF (4x concentr.) Tissuefactor/ 59 sec >600 sec 122 sec 57 sec phospholipids

Taken together, the results of this experiment show that TF and CaCl₂should not be stored mixed together. Thus, separation of TF and CaCl₂into two spatially separated compartments (a) and (b) seems undoubtedlynecessary.

Example 3 Effect of Ellagic Acid/Phospholipids Deposited in the PipetteTip in Wet or Dry Form on Clotting Time

To investigate the effect of an intrinsic activator of coagulation,namely ellagic acid and phospholipids, provided either in dry or in wetform in compartment (b) of the pipette tip (formed by a porous insert asdescribed in Example 1) on clotting time, viscoelastic measurements wereperformed by using the equipment and procedures described in Example 1above.

Results are shown in Table 3 below.

TABLE 3 Clotting times (CT) obtained by identical activator solutionswithout additives after storage as liquid or dried tip for 7 days atroom temperature (tip insert made from polyether, each value wascalculated as average of 4 measurements with human plasma). The wetstorage of ellagic acid results in comparable CT values as the control,but the degradation during dry storage and/or dissolution delay can becompensated for by 35% more activator content and adding 2% sucrose. CTof CT of dry tip Activator CT control dry tip CT of wet tip (1,3xconcentr.) Ellagic acid/ 164 sec 258 sec 162 sec 161 sec phospholipids

Example 4 Effect of CaCl₂ Deposited in the Pipette Tip in Either Wet orDry Form in Compartment (a) or (b) on Clotting Time

To investigate the effect of CaCl₂ deposited either in dry or in wetform in compartment (a) or (b) of the pipette tip, viscoelasticmeasurements were performed by using the procedures, equipment andcompartment specifications as described in Example 1 above.

Results are shown in Table 4 below.

TABLE 4 Clotting times (CT) obtained after storing CaCl₂ in the tip forone week at room temperature (each value was calculated as average of 4measurements with human plasma). No significant differences to thecontrol CT are observed for all four approaches. CaCl₂ CaCl₂ dry in wetin CaCl₂ dry in CaCl₂ wet in compart- compart- CT compartmentcompartment ment ment Activator control (a) (a) (b) (b) Ellagic 194 sec207 sec 205 sec 192 sec 189 sec acid

The invention claimed is:
 1. A pipette tip comprising a constituent (A)and a constituent (B), the pipette tip having: an open upper endconfigured to fit to an end of a pipette; an open lower end configuredto aspirate and release a liquid sample; and a longitudinal axisspanning a length from the open upper end to the open lower end; whereina first region of the pipette tip along the longitudinal axis comprisesconstituent (A) and a second region of the pipette tip along thelongitudinal axis comprises constituent (B), said first region beingselected from the group consisting of a first circumferential reagentlayer deposited on a first circumferential inner surface area of thepipette tip and a first porous insert contained by the firstcircumferential inner surface area of the pipette tip, said secondregion being selected from the group consisting of a secondcircumferential reagent layer deposited on a second circumferentialinner surface area of the pipette tip and a second porous insertcontained by the second circumferential inner surface area of thepipette tip, constituent (A) being physically confined and releasablycoupled to one of the first region and the second region and beingseparate from constituent (B) along the longitudinal axis of the pipettetip, and constituent (B) being physically confined and releasablycoupled to the other one of the first region and the second region andbeing separate from constituent (A) along the longitudinal axis of thepipette tip, wherein: (a) the first circumferential inner surface areaand the second circumferential inner surface area are non-overlappingalong the longitudinal axis of the pipette tip, and (b) constituent (A)is different than constituent (B); and (c) constituent (A) andconstituent (B) are configured to form a diagnostic composition upontheir release from the first region and the second region.
 2. Thepipette tip according to claim 1, wherein at least one of constituents(A) and (B) comprises at least one of the following components (i) and(ii): i) an activator of coagulation; and ii) a calcium salt.
 3. Thepipette tip according to claim 1, wherein each of the constituents (A)and (B) is independently from each other a liquid formulation, anessentially dry formulation, or a dry formulation.
 4. The pipette tipaccording to claim 2, wherein 1) constituent (A) comprises component (i)but not component (ii) and constituent (B) comprises component (ii) butnot component (i); or 2) constituent (A) comprises component (ii) butnot component (i) and constituent (B) comprises component (i) but notcomponent (ii).
 5. The pipette tip according to claim 2, wherein theactivator of coagulation is an extrinsic activator and/or an intrinsicactivator.
 6. The pipette tip according to claim 2, wherein component(i) is an extrinsic activator of coagulation and component (ii) is acalcium salt; or wherein component (i) is an intrinsic activator ofcoagulation and component (ii) is a calcium salt.
 7. The pipette tipaccording to claim 5, wherein the extrinsic activator is a Tissue Factor(TF).
 8. The pipette tip according to claim 5, wherein the intrinsicactivator of coagulation is selected from the group consisting ofcelite, ellagic acid, sulfatit, kaolin, silica, RNA, and mixturesthereof.
 9. The pipette tip according to claim 1, wherein at least oneof constituents (A) and (B) comprises one or more components selectedfrom the group consisting of a coagulation activating factor, acoagulation inhibitor and an active-component inhibitor.
 10. The pipettetip according to claim 9, wherein the active-component inhibitor isselected from one or more platelet inhibitors, fibrinolysis inhibitors,and/or heparin inhibitors.
 11. The pipette tip according to claim 10,wherein the platelet inhibitor is a cytoskeleton inhibitor and/or aGPIIb/IIIa antagonist; and/or the fibrinolysis inhibitor is selectedfrom the group consisting of aprotinin, tranexamic acid, eaca,thrombin-activated fibrinolysis inhibitor, plasminogen activationinhibitor 1/2, α2-antiplasmin, and α2-macroglobulin; and/or the heparininhibitor is selected from the group consisting of heparinase,protamine, protamine-related peptides and their derivatives, andcationic polymers.
 12. The pipette tip according to claim 9, wherein thecoagulation activating factor is selected from the group consisting ofFI, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, and TF.
 13. The pipettetip according to claim 9, wherein the coagulation inhibitor is selectedfrom tissue factor pathway inhibitor, antithrombin I-IV, or activatedprotein C.
 14. The pipette tip according to claim 1, wherein at leastone region selected from the group consisting of the first region andthe second region is a circumferential regent layer, the circumferentialreagent layer being selected from the group consisting of the firstcircumferential reagent layer and the second circumferential reagentlayer.
 15. The pipette tip according to claim 1, wherein at least oneregion selected from the group consisting of the first region and thesecond region is a porous insert, the porous inset being selected fromthe group consisting of the first porous insert and the second porousinsert.
 16. The pipette tip according to claim 1, wherein one regionselected from the group consisting of the first region and the secondregion is a circumferential reagent layer and the other one region is aporous insert, wherein, the circumferential reagent layer is selectedfrom the group consisting of the first circumferential reagent layer andthe second circumferential reagent layer, and the porous insert isselected from the group consisting of the first porous insert and thesecond porous insert.
 17. The pipette tip according to claim 7, whereinthe Tissue Factor (TF) is selected from the group consisting oflipidated TF, rTF, and combinations thereof.
 18. The pipette tipaccording to claim 15, wherein the porous insert has a minimum porediameter of 2 μm and a maximum pore diameter of 2.0 mm.
 19. The pipettetip according to claim 11, wherein the cationic polymer ishexadimethrine bromide (polybrene).